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  • 1.
    Andersson, Sofia
    et al.
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Kuttuva Rajarao, Gunaratna
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Land, Carl Johan
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Dalhammar, Gunnel
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Biofilm formation and interactions of bacterial strains found in wastewater treatment systems2008In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 283, no 1, p. 83-90Article in journal (Refereed)
    Abstract [en]

    Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.

  • 2. Graslund, S.
    et al.
    Larsson, M.
    Falk, R.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hoog, C.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Single-vector three-frame expression systems for affinity-tagged proteins2002In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 215, no 1, p. 139-147Article in journal (Refereed)
    Abstract [en]

    An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.

  • 3. Hansson, M.
    et al.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Nguyen, T. N.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae2002In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 210, no 2, p. 263-270Article in journal (Refereed)
    Abstract [en]

    General expression vectors, designed for intracellular expression or secretion of recombinant proteins in the non-pathogenic Staphylococcus carnosus, were constructed. Both vector systems encode two different affinity tags, an upstream albumin binding protein and a downstream hexahistidyl peptide, and are furnished with cleavage sites for two site-specific proteases for optional affinity tag removal. To evaluate the novel vectors, the gene encoding the outer membrane protein A (OmpA) of Klebsiella pneumoniae was introduced into the vectors. Efficient production was demonstrated in both systems, although, as expected for OmpA fusions, somewhat better intracellularly, and the fusion proteins could be recovered as full-length products by affinity chromatography.

  • 4.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Severa, Denise
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display2008In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 278, no 1, p. 128-136Article in journal (Refereed)
    Abstract [en]

    The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.

  • 5.
    Kuttuva, Gunaratna R.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nekhotiaeva, N.
    Good, L.
    Peptide-mediated delivery of green fluorescent protein into yeasts and bacteria2002In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 215, no 2, p. 267-272Article in journal (Refereed)
    Abstract [en]

    Stringent microbial cell barriers limit the application of many substances in research and therapeutics. Carrier peptides that penetrate or translocate across cell membranes may help overcome this problem. To assess peptide-mediated delivery into two yeast and three bacterial species, a range of cell penetrating and signal peptide sequences were fused to green fluorescent protein (GFP), expressed in Escherichia coli, partially purified and incubated with growing cells. Fluorescence microscopy indicated several peptides that mediated delivery. In particular, VLTNENPFSDP efficiently delivered GFP into Candida albicans and Staphylococcus aureus, while YKKSNNPFSD was most efficient for Bacillus subtilis and CFFKDEL for Escherichia coli. Carrier peptides may improve delivery of certain large molecular mass molecules into microorganisms for research and therapeutic applications.

  • 6. Lehtio, J.
    et al.
    Wernérus, Henrik
    KTH, Superseded Departments, Biotechnology.
    Samuelson, Patrik
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula T.
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain2001In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 195, no 2, p. 197-204Article in journal (Refereed)
    Abstract [en]

    The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells. since full-length proteins could be extracted and affinity-purified. Furthermore. surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization Tor the generation of whole-cell microbial tools Fur different applications will be discussed.

  • 7.
    Löfblom, John
    et al.
    KTH, School of Biotechnology (BIO).
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO).
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO).
    Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display2005In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 248, no 2, p. 189-198Article in journal (Refereed)
    Abstract [en]

    We have investigated a staphylococcal surface display system for its potential future use as a protein library display system ill combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this Study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1: 1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to Surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections. (c) 2005 Federation of European Microbiological Societies.

  • 8. Nguyen, Thu-Ha
    et al.
    Splechtna, Barbara
    Krasteva, Stanimira
    Kneifel, Wolfgang
    Kulbe, Klaus D.
    Divne, Christina
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Haltrich, Dietmar
    Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R222007In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 269, no 1, p. 136-144Article in journal (Refereed)
    Abstract [en]

    beta-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The K-m and V-max values for lactose and oNPG were 4.04 +/- 0.26 mM, 28.8 +/- 0.2 mu mol D-glucose released per min per mg protein, and 0.73 +/- 0.07 mM, 361 +/- 12 mu mol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with K-i,K-s=31.7 +/- 3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. beta-Galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.

  • 9. Nord, O.
    et al.
    Gustrin, A.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Fluorescent detection of beta-lactamase activity in living Escherichia coli cells via esterase supplementation2005In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 242, no 1, p. 73-79Article in journal (Refereed)
    Abstract [en]

    The TEM-1 beta-lactamase protein fragment complementation assay was investigated for its applicability in affinity protein-based interaction studies in Escherichia coli, using an affibody-based model system. Results from co-transformation experiments showed that an ampicillin resistant phenotype was specifically associated with cognate affibody-target pairings. Attempts to monitor P-lactamase complementation in vitro with the fluorescent P-lactamase substrates CCF2/AM and CCF2 showed that E. coli lacks an esterase activity necessary for activation of the esterified and membrane-permeable CCF2/AM form of the substrate. Interestingly, supplementation of the assay reaction with a purified fungal lipase (cutinase) resulted in efficient activation of CCF2/AM in vitro. Further, periplasmic expression of cutinase allowed for fluorescent discrimination between P-lactamase positive and negative living E. coli cells using the CCF2/AM substrate, which should open the way for novel applications for this prokaryotic host in protein interaction studies.

  • 10.
    Wernérus, Henrik
    et al.
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Vector engineering to improve a staphylococcal surface display system2002In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 212, no 1, p. 47-54Article in journal (Refereed)
    Abstract [en]

    A previously developed expression system for surface display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signals from a Staphylococcus hyicus lipase and the cell wall anchoring part of Staphylococcus aureus protein A (SpA) to achieve surface display of expressed recombinant proteins. The system has been successfully used in various applications but the vector has not been considered genetically stable enough to allow protein library display applications, which would be of obvious interest. A new set of vectors, differing in size and devoid of a phage f1 origin of replication, were constructed and evaluated in terms of bacterial growth characteristics and vector stability. Furthermore, surface expression of a model surface protein was monitored by an enzymatic whole-cell assay and flow cytometry. The engineered expression vectors demonstrated dramatically improved stability and growth properties and two of the novel vectors demonstrated retained high surface density of the displayed model protein. The flow cytometry was found to be a powerful toot for observing the surface density of displayed heterologous proteins, and would thus be a rational strategy for monitoring the optimisation of any surface display system. The implications of these improved display vectors for future protein library applications are discussed.

  • 11. Westberg, J.
    et al.
    Persson, Anja
    Pettersson, B.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Johansson, K. E.
    ISMmy1, a novel insertion sequence of Mycoplasma mycoides subsp mycoides small colony type2002In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 208, no 2, p. 207-213Article in journal (Refereed)
    Abstract [en]

    A new insertion sequence, ISMmy1, has been identified in the bovine pathogen Mycoplasma mycoides subsp. mycoides biotype small colony MmymySC. The occurrence of ISMmy1 in 15 MmymySC strains and 12 other mycoplasmas was examined by Southern blotting. All MmymySC strains showed identical hybridisation patterns except for the type strain PG11, the vaccine strain T1Sr49, and the strain Afade, which all had unique patterns. ISMmy1-like sequences were also found in the bovine pathogen Mycoplasma bovis strain Donetta(T) while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy1. This observation suggests horizontal transfer between MmymySC and M. bovis.

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